Technique Optimization In Preparation Of Recombinant Polypeptide CS5931,And Its Inhibitory Effect On Colon Cancer In Vitro And In Vivo

Objective:This study aims to optimize the technique in preparation of recombinant polypeptide CS5931, and its effect on colon cancer in vitro and in vivo, therefore providing a guide for further pilot amplification and pharmacology study.Methods:1. The plasmid p ET28a-CS5931 and p ET28a-DDDK-CS5931 were transformed into Escherichia coli and expressed the recombinant polypeptide CS5931 and CS5931-EK. Prepared the bacterial sample by super centrifugation and sonication.Purified the CS5931 by Ni2+-NTA with AKTA-Purifier 100. Detected the expression of recombinant polypeptides CS5931 and CS5931-EK by SDS-PAGE and Western blot.2. The fermentation process of recombinant CS5931 expression in Escherichia coli BL21(DE3)/p ET28a-CS5931 was studied. The effects of factors such as carbon source、nitrogen source、phosphate、Mg2+、concentration of IPTG、induction temperature and induction time were all investigated. Plackett-Burman and Central-Composite design were developed based on one factor tests.3. By changing the denaturation and renaturation components,including the small molecules substances, such as glycerol, urea and GSH/GSSH, and investigated the expression of recombinant polypeptide CS5931 and its antitumor activity.4. Antitumor effect of expressed CS5931 and CS5931-EK were studied by analysis of cytotoxicity activity of tumor cells in vitro by MTT assay. Via human colon cancer cells HCT116 xenografts in nude mice, the antitumor activity of recombinant polypeptide CS5931 was investigated in vivo.Results:1. The plasmid p ET28a/CS5931 and p ET28a-DDDK-CS5931 were successfully constructed into Escherichia coli and expressed recombinant polypeptide CS5931 and CS5931-EK. The purified CS5931 was a single band with the purification rate 98%. The yield of CS5931 and CS5931-EK were 2.0 mg/L and 1.5 mg/L,respectively.2. The optimized fermentation contained the concentration of yeast extract 30 g/L,the concentration of glycerol 0.45%, the concentration of IPTG 0.75 m M,induction temperature 16 ℃, induction time 20 h. The yield of CS5931 changed to 7.5 mg/L. It was 2.75 times better than before optimizing 2.0 mg/L.3. The results showed that the expressed CS5931 not only before optimizing but also after optimizing all inhibited the growth of human colon carcinoma cells HCT116,with IC50 values of 23.2,11.6 μM,respectively. The peptide activity of refolded CS5931 was enhanced one time and achieved the set. The renaturation components were 50 m M Tris, 0.5 m M EDTA, 50 m M Na Cl, 0.5 m M L-arginine,5% glycerol, 2 M urea and 2 m M/0.2 m M GSH/GSSH.4. Via human colon cancer cells HCT116 xenografts in nude mice, the recombinant polypeptide CS5931 had antitumor activity in vivo. It significantly inhibited the growth of HCT116 colon cancer cells at 25 mg/kg concentration, and the antitumor activity was dose-dependent. The antitumor activity was better than positive control drug cyclophosphamide(CTX) at the same dose(50 mg/kg).While no animal deaths reveal this recombinant polypeptide CS5931 with a certain security.Conclusions:1. The yield of CS5931 increased from 2.0 mg/L to 7.5 mg/L with 2.75 times by one single test, Plackett-Burman and Central-Composite design. The recombinant polypeptide CS5931 has good inhibitory activity on the tumor cells in vivo and in vitro. His-tag had no effect on human colon cancer HCT116 cell.2. Recombinant polypeptide CS5931 can significantly inhibit the proliferation of human colon cancer cells HCT116 in vitro and in vivo, suggesting that CS5931 has potential application prospect in treatment of colon cancer.